Method of screening

Abstract

A method of screening for a molecule which decreases apoptosis of a cell, comprising: i) combining the candidate molecule and an assay cell; and ii) determining the change in survival of the assay cell in the presence of the molecule relative to a control. In one embodiment, the assay cell is treated with an apoptosis inducing agent prior to or between steps i) and ii). In one example, the treating agent reduces the level or activity of a pro-survival member of the Bcl-2 protein family, such as Bcl-x L or Mcl-1. In another embodiment, the level or activity of at least one pro-survival member of the Bcl-2 family is reduced in the cell of step i). In some embodiments, this is independent of any effect of the candidate molecule or apoptosis promoting agent. By reducing the level or activity of one or more pro-survival Bcl-2 protein family members in the assay cell, the cell will undergo apoptosis mediated inter alia by Bak or Bax or Bak and Bax unless it is rescued by the candidate molecule.

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       3 . A method of screening for a molecule which decreases apoptosis of a cell, comprising:
 i) combining a candidate molecule with an assay cell;   ii) determining the change in survival of the assay cell in the presence of the molecule relative to a control; and
 prior to or between steps i) and ii), treating the assay cell to induce apoptosis with an agent which reduces the level and/or activity of a pro-survival member of the Bcl-2 protein family or an agent which enhances the level and/or activity of a pro-apoptosis member of the Bcl-2 protein family. 
   
   
   
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       6 . The method of  claim 3 , wherein agent reduces the level and/or activity of Bcl-x L  and/or Mcl-1. 
   
   
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       8 . The method of  claim 3 , wherein agent enhances the level and/or activity of Bak or Bax or Bak and Bax. 
   
   
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       16 . The method of  claim 3 , wherein the assay cell is modified to enhance its sensitivity to an apoptosis inducing agent. 
   
   
       17 . The method of  claim 16 , wherein the assay cell is genetically modified to reduce the level or activity of at least one pro-survival member of the Bcl-2 family. 
   
   
       18 . The method of  claim 17 , wherein the cell is an Mcl-1 deficient cell. 
   
   
       19 . The method of  claim 18 , wherein the agent reduces the level or activity of Bcl-x L . 
   
   
       20 . The method of  claim 18 , wherein the agent is a BH3 domain mimicking agent. 
   
   
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       24 . The method of  claim 3 , wherein the candidate molecule is a small molecule, inhibitory RNA, antibody, aptamer, peptide, peptidomimetic or constrained peptide. 
   
   
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       26 . The method of  claim 16 , further comprising:
 iii) combining the candidate molecule with a cell and determining the change in survival of the cell in the presence of the molecule relative to a control.   
   
   
       27 . The method of  claim 26 , wherein the cell is a mammalian cell. 
   
   
       28 . The method of  claim 27 , wherein the mammalian cell is a myeloid cell, lymphoid cell, neural cell, epithelial cell, endothelial cell, stem or progenitor cell, hepatocyte, myoblast, osteoblast, osteoclast, lymphocyte, keratinocyte, melanocyte, mesothelial cell, germ cell, muscle cell, fibroblast, a transformed cell, a cancer cell. 
   
   
       29 . The method of  claim 27 , wherein the mammalian cell is a cell subject to enhanced apoptosis in an apoptosis mediated disease or condition. 
   
   
       30 . The method of  claim 3 , further comprising:
 iii) combining the candidate molecule with a cell and determining the change in survival of the cell in the presence of the molecule relative to a control.